Longitudinal Network Changes and Phenoconversion Risk in Isolated REM Sleep Behavior Disorder

Isolated rapid eye movement sleep behavior disorder (iRBD) is a prodromal syndrome for Parkinson’s disease (PD) and related α-synucleinopathies. We conducted a longitudinal imaging study of network changes in iRBD and their relationship to phenoconversion. Expression levels for the PD-related motor and cognitive networks (PDRP and PDCP) were measured at baseline, 2 and 4 years, along with dopamine transporter (DAT) binding. PDRP and PDCP expression increased over time, with higher values in the former network. While abnormal functional connections were identified initially within the PDRP, others bridging the two networks appeared later. A model based on the rates of PDRP progression and putamen dopamine loss predicted phenoconversion within 1.2 years in individuals with iRBD. In aggregate, the data suggest that maladaptive reorganization of brain networks takes place in iRBD years before phenoconversion. Network expression and DAT binding measures can be used together to assess phenoconversion risk in these individuals.


Introduction
Isolated rapid eye movement (REM) sleep behavior disorder (iRBD) is a parasomnia that is characterized by loss of muscle atonia and abnormal behaviors occurring during REM sleep.It is considered a prodromal stage of -synucleinopathies, which include the major Lewy body disorders, i.e., Parkinson's disease (PD) and dementia with Lewy bodies (DLB) 1,2 .Indeed, 60% of iRBD patients develop overt clinical signs of these disorders over 10 years 3,4 .Nevertheless, predicting when an individual with iRBD will develop signs and symptoms of PD or DLB is a major challenge.At a broader level, iRBD provides a unique population in which to assess the e cacy of potential disease-modifying therapies in at-risk individuals.That said, accurate determination of disease progression in these individuals is a daunting task given that characteristic clinical manifestations have yet to appear.
In a previous study with [ 18 F]-uorodeoxyglucose (FDG) positron emission tomography (PET), we found that iRBD is associated with elevated expression of the PD-related motor network (termed PD-related metabolic pattern, PDRP) [5][6][7] .Likewise, reduced dopamine transporter (DAT) binding in the putamen, re ecting presynaptic dopaminergic attrition, has been documented in iRBD and proposed as a potential predictor of phenoconversion 4,8 .In the current longitudinal PET study, we used FDG PET to examine the time course of PDRP expression in iRBD patients.Given that cognitive impairment has been found to be a risk factor for phenoconversion in iRBD, we also evaluated parallel changes in the expression of the PDrelated cognitive network (termed PD-related cognition pattern, PDCP) 5,7,9 in the same individuals.In addition to measuring longitudinal changes in PDRP and PDCP expression in unconverted iRBD subjects, we employed advanced computational techniques to study the gain and loss of speci c metabolic connections linking nodes within and between the two disease topographies 10 , and assess the impact of the connectivity changes on network structure and function.Finally, we used [ 18 F]-uoropropyl-β-CIT (FPCIT) PET to determine the relationship of the network changes to loss of caudate and putamen DAT binding in iRBD, and the complementary role of these measures as predictors of phenoconversion.
Nodal changes in the PDCP space were comparatively less striking.Signi cant declines in local metabolic activity were seen in the inferior parietal lobule (F (2,34) = 7.23, p < 0.004; one-way RMANOVA), but deviations from normal in this region were signi cant only at the third timepoint (p < 0.05 compared to HC1; Student's t-test).That said, metabolic activity at the other major PDCP nodes did not change over time (p > 0.25; one-way RMANOVAs); reductions in these regions did not reach signi cance even at the last imaging timepoint (p > 0.08).

Gain and loss of functional connections
To identify the altered regional interactions between PD network nodes that underlie the differences in network metrics (see Methods), we evaluated the gain and loss of metabolic connectivity at each timepoint with respect to the HC1 subjects.The speci c connections gained in the iRBD group, i.e., those present in iRBD but absent in healthy subjects, for the three timepoints are listed in Table S1A, top and displayed in Fig. 2. At the earliest iRBD timepoint, we noted signi cant gain of connections in the PDRP space, i.e., PDRP-PDRP, preferentially linking metabolically active nodes located in the core zone of the network (Table S1A, red font).At later timepoints, the connections gained tended to link metabolically active core nodes with their underactive counterparts in the network periphery (Table S1A, blue font).These connections involved mainly those linking the supplementary motor area (SMA) with posterior cerebral cortical regions, and the amygdala with the middle frontal gyrus (BA 46).These connections were evident only at the nal timepoint or in the case of the amygdala-prefrontal connection, at both the second and third timepoints.
The PDRP-PDRP connections that were lost, i.e., those that were present in healthy subjects but absent in iRBD are listed in Table S1A, bottom.At early timepoints ( rst timepoint alone or in the rst two timepoints), loss of normal functional connections involved mainly those linking metabolically active nodes such as the putamen and cerebellum, and linking active and underactive nodes in the cerebellar vermis and middle frontal gyrus (BA 46), respectively.At later timepoints (last timepoint alone or in both the second and third timepoints), connectional losses were observed between metabolically active nodes in the thalamus and SMA, and between active and underactive nodes corresponding to the insula and the superior medial frontal gyrus (BA 9).Loss of connections between the metabolically active nodes, cerebellum and insula and between underactive nodes in the precuneus and inferior occipital gyrus (BA 19) were discerned at all three timepoints.From a quantitative standpoint, the gain in intrinsic connections across the whole PDRP space (Fig. 3A, left) was 56% at the initial timepoint, which declined to 42-45% at the second and third timepoints.This contrasted with the stepwise increases in the gained connections that were observed linking PDRP and PDCP nodes (Fig. 3A, middle; Table S1B, top), which ranged from 34% at the rst timepoint to 53% at the last timepoint, and with the low-level gain of intrinsic PDCP connections (< 13%) that was present at each of the three timepoints (Fig. 3A, right; Table S1C, top).Loss of connections was also noted, initially involving those linking PDRP nodes (Fig. 3B, left; Table S1A, bottom) or those linking PDRP with PDCP nodes (Fig. 3B, middle; Table S1B, bottom).At subsequent timepoints, loss increased in connections linking PDRP nodes, but was less pronounced for those linking PDRP with PDCP nodes.As with gain, loss in the PDCP space was minimal (< 11%) at all three timepoints (Fig. 3B, right; Table S1C, bottom).In aggregate, the ndings suggest ongoing remodeling of PDRP functional connectivity beginning at the earliest prodromal stages of the disease process.

Changes in network metrics over time
To evaluate the changes in PD network structure and information ow that occur during the progression of iRBD, we computed key graph metrics for PDRP and PDCP at each timepoint (see Methods).For PDRP, we found that degree centrality (Fig. 4A, left) was marginally elevated at the rst timepoint (P CORR =0.034) but not the second; signi cant increases in this metric were observed, however, at the nal timepoint (P CORR <0.001).Signi cant reduction in the clustering coe cient was noted at the rst and third timepoints (P CORR <0.0001).The characteristic path length was elevated at all three timepoints (P CORR <0.001); the largest values for this metric were observed at the last timepoint.Accordingly, abnormal reductions in PDRP small-worldness (Fig. 4A, middle) were present at all three iRBD timepoints (P CORR <0.0001), with the smallest values at the last timepoint.This suggests a progressive imbalance in information ow through the PDRP network, which begins early in the prodromal period.We additionally measured the assortativity coe cient (Fig. 4A, right), an index of the homogeneity of connections in the network space (see Methods).While this graph metric was normal at the initial iRBD timepoint, values were signi cantly elevated at the latter two timepoints (P CORR <0.0001).Thus, while reduction in PDRP small-worldness is evident early in the course of iRBD, other maladaptive features, notably increases in network assortativity, develop later in the prodromal period.
For PDCP, similar changes were observed over time for the various graph metrics.At baseline, degree centrality (Fig. 4B, left) was normal in the network space, but signi cant elevations in this metric were present at the second and third timepoints (P CORR <0.001).That said, signi cant reductions in the clustering coe cient, increases in characteristic path length (P CORR <0.0001), and consequently, reduced small-worldness (P CORR <0.0001; Fig. 4B, middle) were present at all three timepoints.These abnormalities were largest at the nal timepoint.In contrast to PDRP, assortativity (Fig. 4B, right) did not differ signi cantly from normal at either the rst or second timepoints.This metric was elevated, however, at the last timepoint (P CORR <0.0001).

Prediction of time of phenoconversion in individuals with iRBD Predictions based on baseline PDRP expression
We computed baseline PDRP and PDCP expression in [ 99m Tc]-ethylcysteinate dimer (ECD) single photon emission computerized tomography (SPECT) perfusion scans obtained in 17 iRBD subjects as previously reported (see Methods).For both networks, expression values were abnormally elevated in the iRBD group (PDRP: p < 0.004; PDCP: p < 0.03 compared to HC3; Student's t-tests).Of the iRBD subjects, 12 subsequently developed signs of PD (n = 8) or DLB (n = 4) an average of 4.8 ± 3.9 years (range: 0.4 to 14.0 years) after imaging.A signi cant inverse correlation (r=-0.58,p < 0.05; Pearson correlation) was seen between baseline PDRP expression and the time to conversion (Fig. 5A).An analogous correlation was not observed with baseline PDCP values (r = 0.32, p = 0.32).Furthermore, PDRP expression, along with covariates of age, sex, iRBD duration, and UPDRS motor ratings were collectively entered into a multiple regression model to predict the time to conversion for the 12 phenoconverters (see Methods).Indeed, the relationship between PDRP expression and time to conversion remained signi cant (p < 0.02; partial correlation) after adjusting for the other covariates (Fig. 5B, leverage plot).While time to conversion also correlated with UPDRS motor ratings (p < 0.03) and age (p < 0.02) (Fig. 5C, D), those with sex and iRBD duration were not signi cant (p > 0.5).Indeed, of the 10 iRBD subjects in this cohort who converted to PD or DLB in the seven years after imaging, nine had baseline PDRP scores greater than 0.9.Of the ve non-converters, only two had values in this range.These ndings suggest that people with iRBD who have high PDRP levels have a greater risk of subsequent phenoconversion than their low expression counterparts.

Predictions based on longitudinal imaging data
Over the course of the longitudinal study, 4 of the 13 iRBD participants in the current longitudinal PET cohort (Fig. 6, black circles) converted to PD (Subjects #1-3) or DLB (Subject #4) at 0.1, 3.6, 5.5, and 6.4 years (mean 3.90 years) respectively after the nal imaging timepoint.Based on our prior imaging studies of early PD 11,12 , we found that PDRP expression was elevated above + 1.5 at the time of clinical diagnosis, whereas putamen DAT binding was reduced below 40% of the normal mean value.Using the progression rates for these measures estimated by IGM in the iRBD longitudinal cohort (see Methods), we calculated the time it would take to cross both PD thresholds beginning at the individual's nal preconversion scan.For the four converters, the model predicted times to conversion of 0.5, 2.5, 6.2, and 6.2 years (mean 3.85 years).These estimates were in close agreement with the observed phenoconversion times, with error ranging from − 1.2 to + 0.7 years.Thus, the data suggest that the risk of pheonoconversion in individual iRBD subjects can be accurately estimated using a combination of imaging measures.Individuals with high PDRP levels and low putamen DAT binding are at relatively greater risk.This is evident in the four converters in whom values were nearer to the prede ned "PD zone" (Fig. 6, top left, dark gray rectangle).Of the non-converters (open circles), three subjects had normal values for both measures (bottom right) and were furthest away from the "PD zone."These individuals were considered to be at least risk.The remaining iRBD non-converters had abnormal values for one or the other measure but not both and were considered to be at intermediate risk.

Discussion
In this longitudinal study, we observed progressive increases in PDRP and PDCP expression in iRBD, as well as concurrent declines in caudate and putamen DAT binding.While in the iRBD cohort, progression rates were similar for the two PD networks, expression values for the PDRP were greater than PDCP at each timepoint, a nding consistent with earlier cross-sectional studies 6 .Compared to healthy control subjects, PDRP expression was already elevated at baseline, whereas PDCP values did not reach abnormal levels until the nal imaging timepoint.Likewise, the rate of decline in DAT binding was similar for the caudate and putamen, with relative preservation of nigral dopaminergic input to both structures initially, followed by modest reductions to 80-85% of the normal mean at the nal timepoint.Importantly, four iRBD participants converted to PD/DLB during the subsequent 7 years of clinical follow-up.Thus, our imaging data were mainly shaped by the evolution of distinct PD network topographies during the prodromal phase of the illness.Indeed, network progression in iRBD was accompanied by the formation of abnormal functional connections linking PDRP nodes to one another and to PDCP nodes.These connectivity changes are likely maladaptive as suggested by the appearance of abnormal network con gurations soon after the diagnosis of iRBD.
In PD, increases in PDRP expression have been attributed mainly to changes in cortico-striato-pallidothalamo-cortical motor circuits taking place as a downstream consequence of nigrostriatal dopaminergic degeneration 7,13,14 .That said, the abnormal network elevations in iRBD may also be in uenced by changes involving monoaminergic and/or cholinergic pathways in the brainstem and basal forebrain.Indeed, measurements of local metabolic activity at key network nodes (Fig. S1) suggest that while the earliest regional abnormalities were localized to basal ganglia and cerebellum, PDRP core regions in the ventral thalamus and dorsal pons reach signi cance only the nal timepoint.While longitudinal metabolic changes in cortical PDRP regions were not identi ed, signi cant declines were observed in the inferior parietal PDCP node.It is unclear, however, whether this change was the result of loss of cholinergic afferents to the region or α-synuclein deposition, or both.In the absence of rigorous neuropsychological testing, it is also unclear how metabolic changes in this region relate to concurrent cognitive performance in individuals with iRBD.
That said, the results of the connectivity analysis generally accord with the α-synuclein propagation hypothesis 15 .In early iRBD, gain and loss of connections were observed in the PDRP space, involving mainly those linking the basal ganglia to the brainstem and cerebellum.We note that the initial gain of functional connections was between metabolically active core nodes, with subsequent development of abnormal connections linking core nodes to their less active counterparts in the network periphery.Indeed, a similar pattern of connectional gain was previously reported in a cross-sectional analysis of connectivity changes in PD patients 10 .On the other hand, loss of the normal core-to-core and core-toperiphery connections was observed both early and later on in the course of iRBD.For example, we noted early loss of normal connections linking the cerebellum and contralateral putamen in iRBD, suggesting that this pathway is likely disrupted years before the onset of parkinsonian symptoms.Other potentially relevant connections, such as those linking the thalamus to the paracentral lobule (which includes the SMA) and the amygdala to the parahippocampal gyrus, are lost later in the course of iRBD.How these changes related to the post-conversion phenotype of individuals with iRBD remains to be elucidated.When considered in aggregate, however, the ndings point to dynamic network remodeling in iRBD, beginning in the PDRP core zone and spreading outward over time into the periphery.
Interestingly, during iRBD progression, the abnormal connections that were gained were not limited to those between PDRP nodes.Indeed, a signi cant stepwise increase in abnormal connections linking PDRP to PDCP nodes was noted over time (Fig. 2; Fig. 3A, middle).These connections were predominantly cortico-cortical, initially linking frontal lobe regions, with the subsequent appearance of abnormal connections linking the amygdala and parahippocampal gyrus to the frontal operculum.Over time, abnormal connections become evident linking frontal, temporal, and parietal regions with occipital association cortex.That said, gain and loss of functional connections linking PDCP nodes was of small magnitude compared to those between PDRP nodes or between PDRP and PDCP nodes.
We additionally found that the gain and loss of individual connections likely had a detrimental in uence on the structure and function of these networks.In this regard, we found evidence of reduced smallworldness for both PDRP and PDCP, beginning at the rst timepoint.This change is attributed to the abnormal reductions in connections between nearest neighbors (low clustering coe cient) that were evident in both networks, as well as increases in the average number of edges separating individual nodes from the others (high characteristic path length).These topological changes suggest an imbalance between regional specialization (segregation) and parallel processing (integration), which may reduce the e ciency of information ow through both networks.It is also noteworthy that assortativity, the link-averaged correlation coe cient of degree centrality for pairs of connected nodes in a network [16][17][18] , is abnormally increased in iRBD beginning at the second timepoint for PDRP and the third timepoint for PDCP.This metric captures changes in connectional diversity that render networks more vulnerable to random attacks, fragmentation, and critical transitions [19][20][21] .We have previously described progressive increases in PDRP assortativity in multiple groups of PD patients 22 .The current data point to analogous network changes in iRBD, starting years before the onset of motor symptoms.Taken together, the decline in small-worldness and subsequent increase in assortativity seen in longitudinal iRBD are compatible with progressive compromise of information ow through PD-speci c networks in prodromal stages of the disease.
In addition to the changes in PD network expression, we found that DAT binding declined over time in both the caudate and putamen, at similar rates for the two regions.In that regard, FPCIT PET provides complementary information concerning the evolution of dopaminergic de cits in iRBD and the relationship of these changes to the onset of parkinsonism 23 .While presynaptic dopaminergic de cits, particularly those localized to the putamen, are a recognized risk factor for phenoconversion 12,24 , these measurements, as well as other potential predictors such as severity of motor symptoms, autonomic dysfunction, and cerebrospinal uid analysis, do not accurately foretell subsequent transition to clinically manifest PD 25,26 .That said, the longitudinal PET data presented in this study suggest that changes in PDRP expression and putamen DAT binding, when evaluated together, may predict phenoconversion more accurately than either measure alone.Indeed, in our earlier longitudinal PET studies of newly diagnosed PD patients 11,12 , we found that these individuals typically had PDRP expression values above + 1.5 and reductions in putamen DAT binding below 40% of the normal mean value 11,12,24 .Given estimates of the rate of change in these measures, we used the nal pre-conversion scans of each iRBD participant to calculate the time needed to reach the prespeci ed cut-offs for early PD.While in close agreement with observed phenoconversion times determined independently by movement disorders experts, predictions made based on the model need to be replicated in prospective studies of larger iRBD cohorts.
In this regard, we note that only four of the 13 (31%) longitudinal iRBD subjects converted to PD/DLB over a total of 9.6 years (longitudinal imaging for an average of 4.3 years and follow-up clinical observation for an additional 5.3 years).The relatively small number of phenoconversions in this longitudinal cohort can be explained by the milder initial clinical features in these subjects compared to the 12 of 17 (71%) who phenoconverted in the cross-sectional study.Indeed, on enrollment, the phenoconverters in the former group had shorter mean iRBD duration than their cross-sectional counterparts (3.6 vs. 9.6 years), lower initial motor ratings (2.0 vs. 5.4 points), and longer time from initial imaging to phenoconversion (8.3 vs. 4.8 years).It is therefore likely that the iRBD participants in the longitudinal imaging study were further from phenoconversion at the time of enrollment than their counterparts in other studies.
It is noteworthy that other investigators have identi ed iRBD-related spatial covariance patterns (termed iRBDRPs) in FDG PET scans from iRBD subjects 27,28 , or from de novo PD patients with iRBD (termed PD-RBDRP) 29,30 .PD-RBDRP expression measured in iRBD subjects predicted future phenoconversion to PD in iRBD, although this pattern exhibited signi cant topographical overlap with PDRP.More recently, a somewhat different multivariate mapping technique was applied to [ 99m Tc]-HMPAO SPECT cerebral perfusion scans acquired in iRBD and healthy volunteer subjects 31 .The resulting pattern was reported to be a good predictor of future phenoconversion in individuals with iRBD, although prospective validation of the marker is needed in independent pre-conversion samples.This approach contrasted with that employed in the current study in which we used the FDG PET-based PDRP, a highly reproducible network biomarker of PD, which was additionally validated in [ 99m Tc]-ECD SPECT perfusion scans 32,33 .This approach led to a signi cant correlation between baseline PDRP expression and the time to phenoconversion, although further validation is desirable.It is also worth noting that new methods have recently been developed to measure PD network expression in individual subject scans.For example, dynamic [ 18 F]-FPCIT PET can be used to quantify PDRP and PDCP expression in early phase perfusion scans, while caudate and putamen DAT binding can be measured in the late phase images 34 .Noninvasive measurements of network expression are also possible given that elevations of the resting-state fMRI-based PDRP have been documented in both PD and iRBD 5,35,36 .This approach may prove valuable as a non-invasive, widely accessible means of assessing the risk of phenoconversion in individuals with iRBD.
Given that metabolic pattern expression was found to be a sensitive measure of iRBD progression in our study, we performed a preliminary power/sample-size analysis using the PDRP expression as a progression biomarker in a hypothesized 2-year (2-timepoint), blinded disease-modi cation trial of individuals with iRBD randomized to active treatment versus placebo.The results show that randomizing 114-308 subjects may be su cient to detect reductions in progression rate of 30-50% respectively with 80% power (two-sample Student's t-test; G*Power 3.1).This suggests that PDRP measurements can enhance statistical power in such trials.

Subjects
We conducted a 4-year multi-center longitudinal imaging study of individuals with iRBD (13 men; age: 63.5 ± 8.4 years; baseline Uni ed Parkinson's Disease Rating Scale (UPDRS) motor ratings: 1.5 ± 1.7 (mean ± SD)) who were recruited from the Sleep Disorders Clinics at The Feinstein Institutes for Medical Research (n = 8), University of Pennsylvania (n = 4), and Stanford University (n = 1).These participants were scanned at baseline (1.7 ± 2.0 years from the diagnosis of iRBD) with [ 18 F]-uorodeoxyglucose (FDG) positron emission tomography (PET) to map cerebral glucose metabolism; scanning was repeated 2 years (n = 12) and 4 years (n = 10) later.For each of the subjects, longitudinal FDG PET scans were obtained locally at each site using the GE Advance tomograph at the Feinstein Institutes, Manhasset, NY (n = 8), the Siemens Biograph 40 mCT-S PET/CT at the Hospital of the University of Pennsylvania, Philadelphia, PA (n = 4), and the GE Advance tomograph at Stanford University, Palo Alto, CA (n = 1).Of the iRBD cohort, a subset additionally underwent longitudinal [ 18 F]-uoropropyl-β-CIT (FPCIT) PET at baseline (n = 13), 2 years (n = 10), and 4 years (n = 6) to measure dopamine transporter (DAT) binding in the caudate and putamen at each timepoint.These scans were performed within two months of FDG PET using the tomograph at the Feinstein Institutes.The details of this procedure are reported elsewhere 34 .
The following criteria were used for participant selection.Inclusion: (1) A diagnosis of iRBD by a sleep disorder specialist based on clinical history and polysomnographic con rmation; and (2) Minimum age of 30 years.Exclusion: (1) Known diagnosis of PD or other neurodegenerative disorder; (2) Unequivocal signs of parkinsonism on clinical examination and/or past or current histories of treatment with antiparkinsonian medications; (3) Prior history of stroke; (4) Use of neuroleptics, atypical antipsychotics, and antiemetics within the prior year; (5) Narcolepsy or other sleep disorders including moderate or severe obstructive sleep apnea; (6) Individuals receiving benzodiazepines, anticholinergics, selective serotonin reuptake inhibitors, tricyclic antidepressants, monoamine oxidase inhibitors, or other central nervous system active drugs were excluded if iRBD symptoms began after the introduction of these medications.If the iRBD symptoms predated the initiation of these medications, subjects were enrolled but medications were withheld for at least 12 hours before imaging.The participants were followed clinically for an additional 6.4 ± 2.0 years after the nal imaging timepoint.During the clinical phase of the study, all subjects were evaluated for phenoconversion on an annual basis by movement disorders specialists at the participating sites.In this longitudinal iRBD cohort, four male participants (age: 68.9 ± 12.1 years; baseline UPDRS motor ratings: 2.0 ± 2.2; iRBD duration: 3.6 ± 2.8 years) converted to either PD (n = 3) or DLB (n = 1) at an average of 3.9 ± 2.8 years after the nal imaging timepoint.Study protocols and consent forms were approved by the institutional review boards of the collaborating institutions.Written consent was obtained from each participant after detailed explanation of the procedures.

Longitudinal image analysis
FDG PET: Scans from the iRBD participants and a group of 17 age-and sex-matched healthy control subjects (HC1: 15 men and 2 women; age: 59.9 ± 10.7 years) were realigned, spatially normalized, and smoothed as described elsewhere 37 .Expression levels (subject scores) for the PDRP and PDCP networks were computed in each of the iRBD subjects and timepoints; the resulting values were standardized (z-scored) with respect to the HC1 group.These calculations were conducted using inhouse ScAnVP (software freely available upon request at https://feinsteinneuroscience.org) as described elsewhere 7,38 .
In addition to the network calculations, we analyzed the regional data to assess the changes that occurred over time at key network nodes.This was done by measuring local metabolic activity in spherical volumes-of-interest (VOIs) (radius = 2-5 mm) centered on the peak voxel of each of the key regions 39,40 .The resulting values were then ratio-normalized by the global metabolic rate (GMR) for each scan and the changes over time in the iRBD group were determined for each of the regions.
FPCIT PET: Scans from the subset of the iRBD cohort who were additionally scanned with FPCIT PET (see above) were analyzed at each timepoint as described elsewhere 34 .In each scan, standardized regions-of-interest (ROIs) were placed bilaterally on the caudate nucleus, putamen, and occipital cortex; scans were aligned to baseline such that identical ROI templates were applied to the analysis of the scans from all three timepoints 12 .At each timepoint, we estimated caudate and putamen DAT binding by the striatal-to-occipital ratio (SOR), de ned as (striatum -occipital)/occipital counts in a single 10-min frame, beginning 90 min after tracer injection 12,34 .Averaged right and left DAT binding for the putamen and caudate nucleus were separately calculated for each subject and each timepoint, and compared with analogous values from a separate healthy control group (HC2: 4 men and 6 women; age: 60.0 ± 9.9 years).
At each iRBD timepoint, we compared the PDRP/PDCP scores and regional metabolism at key network nodes, as well as caudate/putamen DAT binding, with corresponding HC values using two-sample Student's t-tests.Longitudinal changes in the imaging measures were assessed with one-way repeated measures analysis of variance (RMANOVA) corrected for multiple comparisons using paired Bonferroni tests.In addition, individual growth models (IGMs) 41,42 were used to estimate progression rates for PDRP and PDCP expression over time in the iRBD longitudinal cohort, as well as for declines in caudate and putamen DAT binding in the same individuals.Relationship between changes in these measures over time were evaluated by computing Bland-Altman correlation coe cients.These analyses were performed using SAS Studio (SAS Institute, Cary, NC) software and considered signi cant for p < 0.05, two-tailed.

Gain and loss of speci c connections
To identify functional connections between network nodes that are altered at each iRBD timepoint, we analyzed all signi cant connection pairs using a method described previously for FDG PET 10,37,40 .In particular, we focused on connections linking: (a) PDRP nodes with other PDRP nodes, (b) PDCP nodes with other PDCP nodes, and (c) PDRP with PDCP nodes in the iRBD subjects at each timepoint, and compared the results to HC1.This approach allows for the identi cation of the connections gained in iRBD but absent in HC1 at each timepoint.Likewise, the loss of connections at each longitudinal timepoint was determined by those present in HC1 but absent in iRBD.To this end, we parcellated the brain into 95 ROIs based on the AAL atlas 43 , and used globally normalized metabolic activity in each region to construct matrices of pairwise correlations.For group-level analysis, we used bootstrap resampling (in-house Matlab script; MATLAB R2020a) to generate 100 samples for each group and timepoint.For each iteration, we computed pairwise nodal Pearson correlations.The median values of the iterates (100 bootstrap correlation estimates) were used to create an adjacency matrix for the network in each group and timepoint.These calculations were performed using the Machine Learning Toolbox in MATLAB R2020a.
By this scheme, the magnitude of the correlation (|r|) provided a measure of connectivity between network nodes for each network and timepoint.For a given pair of nodes, group differences in connectivity were described by the absolute difference (|dr|) in the two correlation coe cients 10,37 .For a connection to be gained in iRBD, we required that the magnitude of the correlation coe cient (|r|) that de ned the associated graphical edge be greater than or equal to 0.6 (p < 0.05; Pearson correlation) in the iRBD group but not in HC, and that the corresponding absolute difference (|dr|) from HC be greater than 0.4.(The latter threshold was determined using the HC graph and permuting the regional labels propensity for nodes to form connections with nodes having similar or different attributes.As a general measure of connectional heterogeneity, assortativity provides a measure of overall network stability 19,22,47,48 .These metrics were computed using the Brain Connectivity Toolbox 45 and an in-house Matlab script (MATLAB R2020a).The results were plotted over a range of connectivity thresholds (r = 0.3 to 0.6, at 0.05 increments, corresponding to graph densities between 25% and 60%) 10,22,37 to demonstrate the consistency of group differences in a given metric over multiple adjacent levels.In this study, the minimum threshold was selected at r = 0.3 (graph density ~ 60%) because below this level, group differences can be obscured by the inclusion of random, non-speci c connections.Likewise, the maximum threshold was selected at r = 0.65 (graph density ~ 25%) because above this level, individual nodes may become disconnected from the rest of the graph and distort the results of group comparisons.For graph analysis, we applied the general linear model to the bootstrapped data obtained across connectivity thresholds, followed by post-hoc Bonferroni tests to evaluate differences in graph metrics between groups and timepoints.These analyses were performed using MATLAB R2023a, and the results were considered signi cant for p < 0.05 (two-tailed), incorporating the Bonferroni correction for multiple comparisons.

Estimation of the time to phenoconversion based on network expression
To understand the relevance of the longitudinal imaging changes to clinical onset, we rst determined whether high PDRP expression in iRBD is associated with greater likelihood of conversion 33 .To explore this possibility, we (R.B.P, J-F.G) obtained clinical follow-up on the 17 iRBD subjects (14 men and 3 women; age: 68.9 ± 4.8 years; baseline UPDRS motor ratings: 4.5 ± 3.3) from the Sleep Disorders Clinic of the Hôpital du Sacré-Coeur de Montréal (Montreal, Quebec, Canada) for whom baseline network imaging values were previously reported 33,49 .These participants underwent perfusion scanning with [ 99m Tc]ethylcysteinate dimer (ECD) single photon emission computerized tomography (SPECT) at a single timepoint 12.8 ± 9.5 years from the diagnosis of iRBD, and were followed clinically for an additional 7.1 ± 5.2 years after imaging.In this observational cohort, 12 subjects (9 men and 3 women; age: 69.9 ± 5.1 years; baseline UPDRS motor ratings: 5.4 ± 2.8; iRBD duration: 9.6 ± 6.7 years) converted to either PD (n = 8) or DLB (n = 4) at an average of 4.8 ± 3.9 years after imaging.PDRP expression was computed in each of the scans, and the resulting values were z-scored with respect to analogous values from 17 age-and sex-matched HC subjects (HC3: 13 men and 4 women; age: 66.6 ± 6.0 years).Linear regression analysis was used to determine whether a signi cant relationship existed between baseline PDRP expression and the time from imaging to phenoconversion.Multiple regression analysis and partial correlation leverage plots were used to assess the signi cance of this correlation after adjusting for individual differences in sex, age, duration of iRBD, and UPDRS motor ratings at the time of imaging.Correlations were considered signi cant for p < 0.05.Metabolic connections gained at each timepoint in the iRBD cohort.PDRP and PDCP nodes (represented respectively by red and purple spheres) are displayed, with the radius of each node proportional to the degree centrality (number of connections) at each timepoint (TP).Signi cant connections gained at each timepoint compared to healthy subjects (Tables S1A-C) are represented by yellow lines, with thickness proportional to the strength of the connections (see Methods).Normal connections between network nodes are represented by cyan lines.

Longitudinal analysis
Gain and loss of functional within and between the PDRP and PDCP networks.(A) Left: At baseline (gray), the majority of gained connections (see Methods) were between PDRP nodes, but the proportion (% total) of PDRP-PDRP connections declined incrementally at the 2-year (green) and 4-year (orange) timepoints.Middle: Over the same time period, stepwise increases in gained connections were observed between PDRP and PDCP nodes (PDRP-PDCP).Right: By contrast, proportionally fewer connections were gained between PDCP nodes (PDCP-PDCP), with declines in this category of connections over time.(B) The percentage of healthy connections that were lost at baseline (see Methods) was similar for PDRP-PDRP (left) and PDRP-PDCP (middle) categories.Over time, however, the proportion of lost connections increased in the former category and declined in the latter.Loss of healthy PDCP-PDCP connections over time (right) was less than for the other connection categories, with no signi cant change over time.

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